Unlike most tissues, cartilage does not self-repair following injury. Cartilage is an avascular tissue made up largely of cartilage specific cells, the chondrocytes, special types of collagen, and proteoglycans. The inability of cartilage to self-repair after injury, disease, or surgery is a major limiting factor in rehabilitation of degrading joint surfaces and injury to meniscal cartilage. Osteoarthritis, the major degenerative disease of weight bearing joint surfaces, is caused by eroding or damaged cartilage surfaces and is present in approximately 25% of the over 50-year-old population. In the US more than 20 million people suffer from osteoarthritis, with annual healthcare costs of more than $8.6 billion. In addition, the cost for cartilage repair from acute joint injury (meniscal lesions, patellar surface damage and chondromalacia) exceeds $1 billion annually. Therefore, new therapeutic approaches are needed to heal lesions of cartilage caused by degeneration or acute trauma.
It has now been found that chondrocytes isolated from articular cartilage respond to compounds which activate the non-proteolytic thrombin cell surface receptor (hereinafter xe2x80x9cNPARxe2x80x9d). For example, chondrocytes express approximately 233,000 thrombin binding sites per cell with apparent affinities of approximately 0.1 nM (3000 sites) and 27 nM (230,000 sites) (Example 1). In addition, the compound TP508, an agonist of the non-proteolytic thrombin receptor, stimulates proliferation of bovine chondrocytes in culture in the presence of thrombin as a co-mitogen (Example 2A) and stimulates by itself the proliferation of rat chondrocytes cultured in three dimensional matrix culture (Example 3A). This same TP508 compound also stimulates proteoglycan synthesis as measured by the incorporation of 35S sulfate in both bovine chondrocytes (Example 2B) and 3-dimensional cultures of rat chondrocytes (Example 3B). These in vitro experiments demonstrate that NPAR agonists can stimulate proliferation and matrix production in chondrocytes isolated from articular cartilage. Additional in vivo experiments demonstrate that delivering TP508 in a sustained release formulation to rabbit trochlear grove cartilage defects which extend into the subchondral bone results in repair of the cartilage defect, including repair of subchondral bone, restoration of a normal cartilage surface and integration of the newly formed cartilage with uninjured cartilage outside of the defect area (Example 5).
Based on the results reported in the prior paragraph, novel methods of stimulating chondrocyte growth in vivo and cartilage repair in a subject and novel delivery methods for delivering pharmaceutical compositions to articular defects to aid in surface repair and to prevent articular degradation are disclosed herein.
The present invention is a method of stimulating cartilage growth, regeneration or repair at a site in a subject where cartilage growth, repair or regeneration is needed. The method comprises the step of administering a therapeutically effective amount of an agonist of the non-proteolytically activated thrombin receptor to the site of injury.
Another embodiment of the present invention is a method of stimulating the proliferation and expansion of chrondrocytes in vitro. The method comprises culturing chrondrocytes in the presence of a stimulating amount of an NPAR agonist.
Sites in need of cartilage growth, repair or regeneration are found in subjects with osteoarthritis. Osteoarthritis or degenerative joint disease is a slowly progressive, irreversible, often monoarticular disease characterized by pain and loss of function. The underlying cause of the pain and debilitation is the cartilage degradation that is one of the major symptoms of the disease. Hyaline cartilage is a flexible tissue that covers the ends of bones and lies between joints such as the knee. It is also found in between the bones along the spine. Cartilage is smooth, allowing stable, flexible movement with minimal friction, but is also resistant to compression and able to distribute applied loads. As osteoarthritis progresses, surfaces of cartilage and exposed underlying bone become irregular. Instead of gliding smoothly, boney joint surfaces rub against each other, resulting in stiffness and pain. Regeneration of damaged cartilage and the growth of new cartilage at these arthritic sites would relieve the pain and restore the loss of function associated with osteoarthritis.
Cartilage damage can also occur from trauma resulting from injury or surgery. Sports injuries are a common cause of cartilage damage, particularly to joints such as the knee. Traumatic injury to cartilage can result in the same type of functional impairment. Therefore, sites in a subject with cartilage that has been damaged by trauma or disease are in need of treatment to restore or promote the growth of cartilage.
Applicants have discovered that compounds which stimulate or activate the non-proteolytically activated thrombin receptor (hereinafter xe2x80x9cNPARxe2x80x9d) can stimulate chondrocytes to proliferate. Chondrocytes are cells which make up about 1% of the volume of cartilage and which replace degraded matrix molecules to maintain the correct volume and mechanical properties of the tissue. Applicants have also found that compounds which stimulate or activate NPAR stimulate proteoglycan synthesis in chondrocytes. Proteoglycan is a major cartilage component. Based on these results, Applicants delivered the NPAR agonist TP508, prepared in a sustained release formulation, to defects in rabbit trochlear grove cartilage and discovered that the peptide stimulated repair of the defect that included formation of new cartilage with a normal cartilage surface. The peptide also stimulated layering and integration of this new cartilage into adjacent, uninjured cartilage and restoration of the subchondral bone. It is concluded that NPAR agonists can induce cartilage growth and repair when administered to sites needing cartilage growth and/or repair.
Compounds which stimulate or activate NPAR are said to be NPAR agonists. NPAR is a high-affinity thrombin receptor present on the surface of most cells. NPAR is largely responsible for high-affinity binding of thrombin, proteolytically inactivated thrombin, and thrombin derived peptides to cells. NPAR agonists and antagonists can compete for the affinity binding with thrombin to cells (see, e.g., Glenn et al., J. Peptide Research 1:65 (1988)). NPAR appears to mediate a number of cellular signals that are initiated by thrombin independent of its proteolytic activity. An example of one such signal is the upregulation of annexin V and other molecules identified by subtractive hybridization (see Sower, et. al., Experimental Cell Research 247:422 (1999)). NPAR is therefore characterized by its high affinity interaction with thrombin at cell surfaces and its activation by proteolytically inactive derivatives of thrombin and thrombin derived peptide agonists as described below. NPAR activation can be assayed based on the ability of its agonists, to stimulate cell proliferation when added to fibroblasts in the presence of submitogenic concentrations of thrombin or molecules that activate protein kinase C as disclosed in U.S. Pat. Nos. 5,352,664 and 5,500,412.
NPAR is to be distinguished from other thrombin binding proteins and the cloned family of proteolytically-activated receptors for thrombin, including the receptors PAR1, PAR2, PAR3 and PAR4. PAR1 possesses a specific thrombin cleavage site that allows thrombin cleavage to expose a new amino-terminus domain that acts as a tethered ligand folding back onto itself inducing its activation (see, Vu, et al., Cell. 64:1057 (1991)). PAR2 has a similar mechanism for activation, but is principally activated by trypsin-like enzymes (see, Zhong, et al, J. Biol. Chem. 267:16975 (1992)). PAR3 also has a similar mechanism of activation and appears to function as a second thrombin receptor in platelets (see, Ishihara, et al., Nature. 386:502 (1997)). PAR4 has been detected in mouse megakaryocytes and studies suggest that it also functions in human platelets (see, Kahn, et al., Nature 394:690 (1998)). In contrast with these PAR receptors, activation of NPAR requires no proteolytic cleavage.
Several lines of evidence indicate that NPAR is distinct from PAR receptors: (1) a population of cells has been isolated that express fully functional PAR1 receptors, but are non-responsive to thrombin due to a defect in the NPAR signal transduction pathway (see, Kim, et al., J. Cell. Physiol. 160:573 (1994)); (2) neutrophils bind 125I thrombin with high affinity and their chemotaxis is stimulated by proteolytically inactivated thrombin or NPAR agonists (see, Ramakrishnan and Carney, Mol. Biol. Cell 4. 1993 (1993)), yet they do not express PAR1 (see Jenkins, et al., J. Cell Sci. 108:3059 (1995)); (3) IIC9 fibroblasts over-express PAR1, but do not bind thrombin with high affinity (see, Kim, D. Ph.D. Dissertation. The University of Texas Medical Branch at Galveston, 1995; and Low, et al., xe2x80x9cCancer Cells 3/Growth Factors and Transformationxe2x80x9d, Cold Spring Harbor Laboratory, New York); and (4) NPAR agonists have distinct effects on gene expression from those of the PAR receptor agonist peptides (see, Sower, et. al., Experimental Cell Research 247: 422 (1999).
One example of an NPAR agonist is a thrombin peptide derivative and physiologically functional equivalents, i.e., a polypeptide with no more than about fifty amino acids, preferably no more than about thirty amino acids and having sufficient homology to the fragment of human thrombin corresponding to prothrombin amino acids 508-530 (SEQ ID NO: 5) that the polypeptide activates NPAR. The thrombin peptide derivatives described herein preferably have between about 12 and 23 amino acids, more preferably between about 19 and 23 amino acids. One example of a thrombin peptide derivative comprises a moiety represented by Structural Formula (I):
Asp-Ala-Rxe2x80x83xe2x80x83(I)
R is a serine esterase conserved domain. Serine esterases, e.g., trypsin, thrombin, chymotrypsin and the like, have a region that is highly conserved. xe2x80x9cSerine esterase conserved domainxe2x80x9d refers to a polypeptide having the amino acid sequence of one of these conserved regions or is sufficiently homologous to one of these conserved regions such that the thrombin peptide derivative retains NPAR activating ability.
In one embodiment, the serine esterase conserved sequence has the amino acid sequence of SEQ ID NO: 1 (Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val) or a C-terminal truncated fragment of a polypeptide having the amino acid sequence of SEQ ID NO: 1. It is understood, however, that zero, one, two or three amino acids in the serine esterase conserved sequence can differ from the corresponding amino acid in SEQ ID NO: 1. Preferably, the amino acids in the serine esterase conserved sequence which differ from the corresponding amino acid in SEQ ID NO: 1 are conservative substitutions, and are more preferably highly conservative substitutions. A xe2x80x9cC-terminal truncated fragmentxe2x80x9d refers to a fragment remaining after removing an amino acid or block of amino acids from the C-terminus, said fragment having at least six and more preferably at least nine amino acids.
More preferably, the serine esterase conserved sequence has the amino acid sequence of SEQ ID NO: 2 (Cys-X1-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val; X1 is Glu or Gln and X2 is Phe, Met, Leu, His or Val) or a C-terminal truncated fragment thereof having at least six amino acids, preferably at least nine amino acids.
In a preferred embodiment, the thrombin peptide derivative comprises a serine esterase conserved sequence and a polypeptide having a more specific thrombin amino acid sequence
In a preferred embodiment, the thrombin peptide derivative comprises a serine esterase conserved sequence and a polypeptide having a more specific thrombin amino Arg-Gly-Asp-Ala (SEQ ID NO: 3). One example of a thrombin peptide derivative of this type comprises Arg-Gly-Asp-Ala-Cys-X1-Gly-Asp-Ser-Gly-Gly-Pro-X2-Val (SEQ ID NO: 4). X1 and X2 are as defined above. When the thrombin peptide derivative comprises SEQ ID NO: 4, it preferably has the amino acid sequence of SEQ ID NO: 5 (Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val) or an N-terminal truncated fragment thereof, provided that zero, one, two or three amino acids at positions 1-9 in the thrombin peptide derivative differ from the amino acid at the corresponding position of SEQ ID NO: 5. Preferably, the amino acids in the thrombin peptide derivative which differ from the corresponding amino acid in SEQ ID NO: 5 are conservative substitutions, and are more preferably highly conservative substitutions. An xe2x80x9cN-terminal truncated fragmentxe2x80x9d refers to a fragment remaining after removing an amino acid or block of amino acids from the N-terminus, preferably a block of no more than six amino acids, more preferably a block of no more than three amino acids.
TP508 is an example of a thrombin peptide derivative and has the amino acid sequence of SEQ ID NO: 5. A physiologically functional equivalent of SEQ ID NO: 5 is SEQ ID NO: 6 which has the identical amino acid sequence of SEQ ID NO: 5 and also contains a C-terminal amide.
A xe2x80x9cconservative substitutionxe2x80x9d is the replacement of an amino acid with another amino acid that has the same net electronic charge and approximately the same size and shape. Amino acids with aliphatic or substituted aliphatic amino acid side chains have approximately the same size when the total number carbon and heteroatoms in their side chains differs by no more than about four. They have approximately the same shape when the number of branches in the their side chains differs by no more than one. Amino acids with phenyl or substituted phenyl groups in their side chains are considered to have about the same size and shape. Listed below are five groups of amino acids. Replacing an amino acid in a polypeptide with another amino acid from the same group results in a conservative substitution:
Group I: glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, and non-naturally occurring amino acids with C1-C4 aliphatic or C1-C4 hydroxyl substituted aliphatic side chains (straight chained or monobranched).
Group II: glutamic acid, aspartic acid and non-naturally occurring amino acids with carboxylic acid substituted C1-C4 aliphatic side chains (unbranched or one branch point).
Group III: Lysine, ornithine, arginine and non-naturally occurring amino acids with amine or guanidino substituted C1-C4 aliphatic side chains (unbranched or one branch point).
Group IV: glutamine, asparagine and non-naturally occurring amino acids with amide, substituted C1-C4 aliphatic side chains (unbranched or one branch point).
Group V: phenylalanine, phenylglycine, tyrosine and tryptophan.
A xe2x80x9chighly conservative substitutionxe2x80x9d is the replacement of an amino acid with another amino acid that has the same functional group in the side chain and nearly the same size and shape. Amino acids with aliphatic or substituted aliphatic amino acid side chains have nearly the same size when the total number carbon and heteroatoms in their side chains differs by no more than two. They have nearly the same shape when they have the same number of branches in the their side chains. Example of highly conservative substitutions include valine for leucine, threonine for serine, aspartic acid for glutamic acid and phenylglycine for phenylalanine. Examples of substitutions which are not highly conservative include alanine for valine, alanine for serine and aspartic acid for serine.
Other NPAR agonists include small organic molecules which bind and activate NPAR. Agonists of this type can be conveniently identified with high through-put screening, e.g., with assays that assess the ability of molecules to stimulate cell proliferation when added to fibroblasts in the presence of submitogenic concentrations of thrombin or molecules that activate protein kinase C or with assays that assess the ability of these molecules to compete with 125I-thrombin to cells with surface NPAR receptors, as disclosed in Glenn et al., supra, U.S. Pat. Nos. 5,352,664 and 5,500,412. The entire teachings for Glenn et al., and U.S. Pat. Nos. 5,352,664 and 5,500,412 are incorporated herein by reference.
The term xe2x80x9cNPAR agonistxe2x80x9d also includes compounds and combinations of compounds known to activate NPAR. Examples are disclosed in U.S. Pat. Nos. 5,352,664 and 5,500,412 and include thrombin and DIP-alpha-thrombin.
NPAR agonists used in the method of the present invention are typically administered as one component in a pharmaceutical composition to the site in need of cartilage growth, repair or regeneration. Administering to the site in need of treatment means that the pharmaceutical composition containing the NPAR agonist is administered in sufficient proximity to the site in need of treatment so that cartilage growth or cartilage regeneration occurs at the site (e.g., a greater amount of cartilage growth or better quality of cartilage growth in the presence of the NPAR agonist than in its absence).
In one means of administration, the pharmaceutical composition is a solution comprising the NPAR agonist and a suitable carrier. The solution is applied directly to or in near proximity to the site in need of treatment. Administration of the solution can be conveniently accomplished, for example, intraarticularly by syringe, in close proximity to the damaged tissue by syringe or through a surgical opening. Standard pharmaceutical formulation techniques may be employed such as those described in Remington""s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Suitable pharmaceutical carriers for include, for example, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank""s solution, Ringer""s-lactate and the like.
In another means of administration, the pharmaceutical composition comprises the NPAR agonist and an implantable biocompatible carrier. A biocompatible carrier should be non-toxic, non-inflammatory, non-immunogenic and devoid of other undesired reactions at the implantation site. Suitable carriers also provide for release of the active ingredient and preferably for a slow, sustained release over time at the implantation site.
A number of synthetic biodegradable polymers can serve as carriers with sustained release characteristics. Examples of these polymers include poly xcex1-hydroxy esters such as polylactic acid/polyglycolic acid copolymers and polyanhydrides.
Polylactic acid/polyglycolic acid (PLGA) homo and copolymers are well known in the art as sustained release vehicles. The rate of release can be adjusted by the skilled artisan by variation of polylactic acid to polyglycolic acid ratio and the molecular weight of the polymer (see Anderson, et al., Adv. Drug Deliv. Rev. 28:5 (1997), the entire teachings of which are incorporated herein by reference). The incorporation of poly(ethylene glycol) into the polymer blend allows further attenuation of the release profile of the active ingredient (see Cleek et al., J. Control Release 48:259 (1997), the entire teachings of which are incorporated herein by reference). Suitable implantable PLGA polymers for use as carriers for cartilage growth factors are described in U.S. Pat. Nos. 6,013,853, 5,607,474 and 5,876,452, the entire teachings of which are incorporated herein by reference.
Polyanhydridcs, shown in Structural Formula (II), have well defined degradation and release characteristics that can be controlled by including varying amounts of hydrophobic or hydrophilic monomers such as sebacic acid and 1,3-bis(p-carboxyphenoxy)propane (see Leong et al., J. Biomed. Mater. Res. 19:941 (1985), the entire teachings of which are incorporated herein by reference). To improve mechanical strength, anhydrides are often copolymerized with imides to form polyanhydride-co-imides. Examples of polyanhydride-co-imides that are suitable for orthopaedic applications are poly(trimellitylimido-glycine-co-1,6-bis(carboxyphenoxy)hexane and pyromellityimidoalanine:1,6-bis(p-carboxyphenoxy)hexane copolymers. 
The pharmaceutical compositions can be shaped as desired in anticipation of surgery or shaped by the physician or technician during surgery. It is preferred to shape the matrix to span a tissue defect and to take the desired form of the new tissue. In the case of cartilage repair of large defects, it is desirable to use dimensions that span the defect. After implantation, the material is slowly absorbed by the body and is replaced by cartilage in the shape of or very nearly the shape of the implant.
In one aspect, the carrier is a porous matrix into which progenitor cells may migrate. Cells can often attach to such porous matrices, which can then serve as a scaffolding for tissue growth and thereby accelerate the rate of bone growth. Chondrocytes can be applied to such matrices prior to implant to further accelerate healing. Collagen or a collagen gel is an example of a suitable porous matrix.
In another aspect, the carrier is a viscous solution or gel that is injectable intraarticuarly or at the site in need of treatment. Hyaluronic acid is an example of a carrier of this type. Hyaluronic acid products are commercially available and include ORTHOVISC developed by Anika, SYNVISC, developed by Biomatrix, HYALGAN, developed by Fidia and ARTZ, developed by Seikagaku. Pluronic gel is another example of this type of carrier. Pluronic gels are nontxoic block copolymers of ethylene oxide and propylene oxide. They exhibit thermosetting properties that allow them to exist as viscous liquids at room temperatures, but as gels at body temperatures. Injectable compositions can be applied directly to the site in need of treatment without the need for invasive surgery. Polymers of poly(ethylene oxide) and copolymers of ethylene and propylene oxide are also suitable as injectable matrices (see Cao et al., J. Biomater. Sci 9:475 (1998) and Sims et al., Plast Reconstr.Surg. 98:843 (196), the entire teachings of which are incorporated herein by reference).
A xe2x80x9ctherapeutically effective amountxe2x80x9d is the quantity of NPAR agonist (or chondrocytes) which results in greater cartilage growth or repair in the presence of the NPAR agonist than in its absence. Alternatively or addition, a xe2x80x9ctherapeutically effective amountxe2x80x9d is the quantity of NPAR agonist (or chondrocytes) which results in alleviation of the pain and/or lack of function associated with the cartilage damage. Typically, the agonist (or chondrocytes) is administered for a sufficient period of time to achieve the desired therapeutic or effect. The amount administered will depend on the amount of cartilage growth that is desired, the health, size, weight, age and sex of the subject and the release characteristics of the pharmaceutical formulation. Typically, between about 0.1 xcexcg per day and about 1 mg per day of NPAR agonist (preferably between about 5 xcexcg per day and about 100 xcexcg per day) is administered by continuous release or by direct application to the site in need of carilage growth or repair.
A xe2x80x9csubjectxe2x80x9d is preferably a human, but can also be an animal in need of treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horses and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like).
NPAR agonists can be used to accelerate the growth or to maintain the functionality of isolated chondrocytes. In one embodiment, NPAR agonists can be added to tissue culture medium to stimulate proliferation and provide for more rapid proliferation and/or to prevent apoptotic death or senescence of cells often encountered when primary cell isolates are place in culture. In another embodiment, because the NPAR agonists appear to stimulate matrix production, such NPAR agonists could be used to maintain the differentiated functionality of chondrocytes in culture. NPAR agonists can be used alone in standard defined tissue culture medium or as a supplement to tissue culture medium containing serum or other growth factor to provide additive or synergistic effects or the in vitro production or maintenance of chondrocytes. A sufficient quantity of the NPAR agonist is added to the culture to provide more rapid growth or to maintain greater functionality of the chondrocytes than in the absence of the agonist, i.e., a xe2x80x9cstimulatory amountxe2x80x9d. Typically, between about 0.1 xcexcg/ml and about 100 xcexcg/ml of NPAR agonist is used.
Chondrocytes cultured in the presence of an NPAR agonists can also be used to treat cartilage damage by administering a therapeutically effective amount of the chondrocytes to the site in need of treatment. With respect to chondrocytes, xe2x80x9ctherapeutically effectivexe2x80x9d also means which results in greater cartilage growth or repair with the treatment than in its absence. The administration of chondrocytes to treat cartilage damage is described in U.S. Pat. No. 4,846,835, the entire teachings of which are incorporated herein by reference.
Thrombin peptide derivatives can be synthesized by solid phase peptide synthesis (e.g., BOC or FMOC) method, by solution phase synthesis, or by other suitable techniques including combinations of the foregoing methods. The BOC and FMOC methods, which are established and widely used, are described in Merrifield, J. Am. Chem. Soc. 88:2149 (1963); Meienhofer, Hormonal Proteins and Peptides, C. H. Li, Ed., Academic Press, 1983, pp. 48-267; and Barany and Merrifield, in The Peptides, E. Gross and J. Meienhofer, Eds., Academic Press, New York, 1980, pp. 3-285. Methods of solid phase peptide synthesis are described in Merrifield, R. B., Science, 232: 341 (1986); Carpino, L. A. and Han, G. Y., J. Org. Chem., 37: 3404 (1972); and Gauspohl, H. et al., Synthesis, 5: 315 (1992)). The teachings of these six articles are incorporated herein by reference in their entirety.
The invention is illustrated by the following examples which are not intended to be limiting in any way.
Details of Experiments
Chondrocytes are the primary cell type found in cartilage. In cartilage these cells are normally quiescent, or non-proliferative, and have relatively low metabolic rates. Following injury to cartilage these cells do not readily participate in the repair process. Due to the avascular nature of cartilage, these cells presumably would not see thrombin as an initiator of the repair process.